Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Inhalation of β2-adrenergic receptor (β2AR) agonists in the treatment of asthma is the most effective approach to acutely relieve bronchospasm, reflecting the ability of these agents to dilate the airways by relaxing the surrounding airway smooth muscle (ASM). Chronic use of long-acting β2AR agonists, however, has been associated with heightened bronchoconstrictor responsiveness to airway spasmogens, exacerbation of asthma symptoms, and an increased incidence of asthma-related morbidity and mortality (4,31,37). This aggravation of the asthmatic condition is thought to result from heightened desensitization of the airways to the bronchodilatory action of β2AR agonists, a phenomenon that is exhibited in isolated asthmatic ASM tissues, together with an increased contractile responsiveness of the tissues to bronchoconstrictor agents (2,3,13). Similarly, naïve ASM tissues and cultured ASM cells exposed to a variety of pro-asthmatic stimuli including atopic asthmatic serum, inflammatory cytokines, and inoculation with rhinovirus, also display attenuated β2AR-mediated relaxation accompanying reduced cAMP generation and increased ASM constrictor responsiveness (20,39). To date, investigations into the etiology of the tolerance of asthmatic airways to β2AR stimulation have largely focused on the potential contributions by mechanisms implicated in mediating homologous (agonist-specific) and/or heterologous (non-agonist-specific) desensitization of the β2AR in ASM (12,39). Accordingly, the role played by phosphorylation of the β2AR by G protein-coupled receptor (GPCR) kinases (GRKs) and cAMP-dependent protein kinase A (PKA) in mediating homologous β2AR desensitization has been demonstrated acutely following exposure of ASM cells to β2AR agonists, and that played by PKA activation in mediating heterologous β2AR desensitization has been demonstrated following more prolonged exposure of ASM to other receptor-coupled or non-receptor-coupled cAMP-elevating agents (12,15,35,39). Collectively, these studies have provided important information regarding the mechanisms involved in uncoupling of the β2AR from its associated Gs protein-mediated accumulation of cAMP, a process resulting in impaired ASM relaxation in the β2AR-desensitized state. These mechanisms notwithstanding, a host of studies conducted on different cell types have demonstrated that the attenuated physiological responses to cAMP-elevating agents detected under conditions associated with homologous or heterologous β2AR desensitization are critically regulated by cAMP phosphodiesterase (PDE) activity (6,9,22,42). Moreover, there is compelling recent evidence that PDE activity plays a crucial role in regulating ASM contractility (33), and in mediating the constrictor hyperresponsiveness of the airways accompanying allergen challenge in asthmatic subjects (43) and in animal models of allergic asthma (8,21,24,41). Little is known, however, regarding the mechanism regulating PDE expression and its role in contributing to the impaired cAMP signaling exhibited in ASM under conditions of prolonged homologous or heterologous β2AR desensitization.
Among the PDE superfamily, isoforms of PDE4 account for most of the cAMP hydrolyzing activity in smooth muscle cells, and PDE4 activity has been importantly implicated in regulating ASM contractility (33). The PDE4 family is encoded by four distinct genes (PDE4A-D) that generate multiple PDE4 enzyme variants through activation of different promoters or alternative splicing. (9,22). The dominant PDE4 type expressed in both vascular smooth muscle and ASM cells is PDE4D and, via alternate promoters, PDE4D can encode six “long” isoforms (PDE4D3-5 and PDE4D7-9) and two PDE4D “short” isoforms (PDE4D1-2) (9,22). Cellular expression of PDE4D is regulated by PKA, and the promoter driving transcription of the functionally dominant long isoform, PDE4D5, in ASM cells contains a cAMP response element (CRE) (5,30). In vascular smooth muscle cells, PDE4D expression was also found to be translationally regulated by PKA, as well as by the MAPK, ERK1/2 (32). Moreover, PKA and ERK1/2 were shown to directly regulate PDE4D catalytic activity (9,22).